HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

SEPARATION MODES IN HPLC Normal Phase Stationary and Mobile Phases: In normal-phase HPLC the stationary phase is a polar adsorbent such as bare silica or silica to which polar nonionic functional groups-alcoholic hydroxyl, nitro, cyano (nitrile), or amino-have been chemically attached (e.g., R = -CH2CH2CH2NH2, aminopropyl). Mobile phase: - a nonpolar solvent, such as hexane, to which is added a more polar modifier, such as methylene chloride, to control solvent strength and selectivity. Applications of Normal-Phase HPLC: Normal-phase HPLC is best applied to the separation of compounds that are highly soluble in organic solvents, such as fat-soluble vitamins, or suffer from low stability in aqueous mobile phases, such as phospholipids. Reversed Phase (70 percent of all HPLC separations). Stationary and Mobile Phases: Uses a nonpolar stationary phase (chemically bonded phases) and a polar mobile phase. Many silica-based reversed-phase columns are commercially available, and differences in their chromatographic behavior result from variation in the following: 1. Type of organic group bonded to the silica matrix, such as C18 versus phenyl; 2. Chain length of organic moiety, such as C8 versus C18; 3. Amount of organic moiety per unit volume of packing; 4. Support particle size and shape; 5. Matrix surface area and porosity; 6. Bonded-phase surface topology, such as monomeric versus polymeric; and 7. Concentration of free silanols. Mobile Phase: Reversed-phase HPLC utilizes polar mobile phases, usually water mixed with methanol, acetonitrile, or tetrahydrofuran. Solutes are retained due to hydrophobic interactions with the nonpolar stationary phase and are eluted in order of increasing hydrophobicity (decreasing polarity).

Applications of Reversed-Phase HPLC: Many - for example - plant proteins, water- and fatsoluble vitamins, carbohydrates, soft drinks (caffeine, aspartame, etc.), lipids (including triglycerides and cholesterol, chlorophylls, carotenoids, and anthocyanins) History of HPLC Prior to the 1970's, few reliable chromatographic methods were commercially available to the laboratory scientist. During 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thinlayer chromatography. However, these chromatographic techniques were inadequate for quantification of compounds and resolution between similar compounds. During this time, pressure liquid chromatography began to be used to decrease flow through time, thus reducing purification times of compounds being isolated by column chromatography. However, flow rates were inconsistent, and the question of whether it was better to have constant flow rate or constant pressure was debated. High pressure liquid chromatography was developed in the mid-1970's and quickly improved with the development of column packing materials and the additional convenience of on-line detectors. In the late 1970's, new methods including reverse phase liquid chromatography allowed for improved separation between very similar compounds. By the 1980's HPLC was commonly used for the separation of chemical compounds. New techniques improved separation, identification, purification and quantification far above the previous techniques. Computers and automation added to the convenience of HPLC. Improvements in type of columns and thus reproducibility were made as such terms as microcolumn, affinity columns, and Fast HPLC began to immerge. Although HPLC is widely considered to be a technique mainly for biotechnological, biomedical, and biochemical research as well as for the pharmaceutical industry, these fields currently comprise only about 50% of HPLC users. Currently HPLC is used by a variety of fields including cosmetics, energy, food, and environmental industries. Stationary Phase The stationary phase in HPLC refers to the solid support contained within the column over which the mobile phase continuously flows. The sample solution is injected into the mobile phase of the assay through the injector port. As the sample solution flows with the mobile phase through the stationary phase, the components of that solution will migrate according to the non-covalent interactions of the compounds with the stationary phase. The chemical interactions of the stationary phase and the sample with the mobile phase, determines the degree of migration and separation of the components contained in the sample. For example, those samples, which have stronger interactions with the stationary phase than with the mobile phase, will elute from the column less quickly, and thus have a longer retention time, while the reverse is also true. Normal Phase columns operate on the basis of hydrophilicity by using a polar stationary phase and a less polar mobile phase. Thus hydrophobic compounds elute more quickly than do hydrophilic compounds. Reverse Phase operates on the basis of hydrophobicity by using a stationary phase that consists of silica-based packings with n-alkyl chains covalently bound to them. For example, C-8 signifies an octyl chain and C-18 an octadecyl ligand in the matrix. The more hydrophobic the matrix on each ligand, the greater is the tendancy of the column to retain hydrophobic compounds. Thus hydrophilic compounds elute more quickly than do hydrophobic compounds.

Mobile Phase The mobile phase in HPLC refers to the solvent being continuously applied to the column, or stationary phase. The mobile phase acts as a carrier for the sample solution. A sample solution is injected into the mobile phase of an assay through the injector port. As a sample solution flows through a column with the mobile phase, the components of that solution migrate according to the non-covalent interactions of the compound with the column. The chemical interactions of the mobile phase and sample, with the column, determine the degree of migration and separation of components contained in the sample. For example, those samples, which have stronger interactions with the mobile phase than with the stationary phase, will elute from the column faster, and thus have a shorter retention time, while the reverse is also true. The mobile phase can be altered in order to manipulate the interactions of the sample and the stationary phase. Mobile phase can be run as a single or mixed solvent running at a constant ratio and flow rate (isocratic) or two separate mobile phases can be pumped simultaneously at varying concentrations to facilitate washing compounds from the stationary phase (gradient). Laboratory Procedure: You will be analyzing the samples you isolated from solid phase extraction using HPLC. Anthocyanins exist in plants as glycosides meaning that there are simple sugars attached to the compounds such as glucose. These sugars may (aglycone) or may not (glycoside) be removed by acid hydrolysis (boiling sample in 2N HCl for ~20-60 minutes). In our case, we will conduct an acid hydrolysis using hydrochloric acid dissolved in both methanol and water. We will be using a reversed phase HPLC to separate the 5 major anthocyanin aglycones present in muscadine grapes. These compounds include (in order of elution) delphidin, cyanidin, petunidin, peonidin, and malvidin. The samples that were prepared are dissolved in both water and methanol and you will inject each to determine if any differences are apparent in their chromatographic separation.

Discussion Questions: 1. Why do the compounds elute in the order that they do? 2. How would the compounds elute if the column were normal phase? 3. If the retention time of a compound that had absolutely NO affinity to the column was 1.25 minutes and the flow rate was 0.8 mL/min, what is the total volume of mobile phase contained in the column, tubing, and pumps? Are you surprised at this numberwhy or why not? 4. Why is the solvent that the compounds are dissolved in so important for good separation by HPLC on a reversed phase column? 5. What would the chromatograph look like if we injected 40L instead of 20L of each sample? 6. What would the chromatograph look like if I switched the order of mobile phases? The following this the solvent program we will be using. It consists of 2 mobile phases mixed together by the pumps, which is called a gradient separation. (Using only one solvent is an isocratic separation). Mobile Phase 1: 60% Methanol and 40% Water (pH 2.4) Mobile Phase 2: 100% Water (pH 2.4) Time 0 3 5 10 15 17 end % Phase 1 0 30 50 70 80 100 %Phase 2 100 70 50 30 20 0

The following is a typical HPLC chromatograph of from muscadine grapes.

Mobile Phase 1 = 60:40 (Methanol:Water) Mobile Phase 2 = 100% Water Solvent Flow 1.0 mL/min Sample injected (20 L). Mobile phase is 0% A and 100% B. All compounds stick nicely to the non-polar column, since the mobile phase running is primarily water. t=0 5 minute into the run. Mobile phasea are at 50%, which is causing some separation of the compounds. t=5 8 minutes into the run. Mobile phase 1 is at 62% and compounds are completely separated by now in the column. Elution is only a matter of time.

t=8 11 minutes into the run. The first 2 compounds (Dp and Cy) have eluted. Elution of remaining comounds will follow soon, elution order is now set. t=11